Xcelligence rtca dp system

Cell migration experiments were conducted in the xCELLigence RTCA DP system (Roche). The cells were suspended in serum-free medium and seeded in the upper chambers of 16-well CIM-Plate 16 plates (40,000 cells in 150 μl medium/well; Roche). Regular medium with 10% FBS was added to the lower chamber of the CIM-Plate 16. The experiment setting and plate design were similar to those of conventional Transwell migration assays. The cell index, which is proportional to the number of cells that migrate through the pores of the upper chamber, was recorded in real-time every 30 min for up to 24 h. The average cell migration index was calculated from at least four replicate experiments, and is presented as mean ± SE.

HMSC and MG63 monolayer cultures (50,000/well) were cultured on E-plates (OLS, Bremen, Germany) and real time proliferation was measured for 100 h after administration of the maximum therapeutic plasma concentration of modafinil (11.2 µg/mL), atomoxetine (0.9 µg/mL) and guanfacine (17.7 g/mL) by an xCELLigence RTCA DP system (Roche, Basel, Switzerland). The real-time proliferation measurement was performed by cell contacts with gold electrodes at the well bottom, which induce an increase of impedance.

The cell migration and invasion experiments were performed using the xCELLigence RTCA DP system (Roche, Mannheim, Germany) as described previously.^{23 (link), 50 (link)} Briefly, cells serum-starved for 6 h were resuspended with serum-free medium and seed into up chamber of CIM-plate 16 with or without precoated Matrigel (1:40 dilution), which the low chamber was added medium containing 5% FBS, for real-time cell analysis by using the RTCA DP Instrument. The increase of the impedance correlates with increasing numbers of migrated cells were recorded every 15 min and the cell index Slope (1/hour) for a time period/range was calculated according to RTCA Software Manual (Version 1.2).

Cell migration was dynamically recorded using the xCELLigence RTCA-DP system (Roche, Germany) following the manufacturer instructions. 25, 000 cells were plated in the upper chamber coated with collagen-type I and the migration into the bottom chamber, in response to fetal serum as a chemoattractant, was monitored during 24 hours. The electrical impedance caused by the migration of the cells from the upper chamber to the lower chamber is converted to Cell Index Value. Thus, the Cell Index is an indicator of the cell capacity to invade the matrix and migrate. The RTCA Software 1.2 was used to analyze the data.

Wound migration of cells was measured using Culture-Inserts (Ibidi GmbH, Martinsried, Germany). The Culture-Inserts were placed in 30 mm dish, and siRNA-control and siRNA-hYSK1-transfected SK-EML-28 cells were seeded at a density of 5 × 10⁴ cells in each well with the Culture-Inserts. After 24 h of incubation, the Culture-Inserts were removed, a cell-free gap of 500 μm was created and then transferred to a hypoxic (1% CO₂) chamber. The movement of cells was detected by an inverted microscope (BX50, Olympus, Tokyo, Japan) by time course.
Cell movement was analyzed using AVI meta imaging software. For real-time cell migration, we used the xCELLigence RTCA DP system and fibronectin-coated CIM plates (Roche Diagnostics, Basel, Switzerland). Cells were seeded at 10,000 cells per well and the migratory behavior of each cell line was monitored for 30 h. The assay was performed based on the manufacturer’s instructions (Roche Diagnostics, Basel, Switzerland).

Cell proliferation assays were performed using the xCELLigence RTCA DP system (Roche, Mannheim, Germany). A cell density of 1 × 10³ (MDA-MB231), 4 × 10⁴ (MCF-7, MCs) or 8 × 10⁴ MG cells per well were plated and proliferation/morphology was analyzed for 48 h in quadruplets. Treatment with 5A1 was performed once at time point 0 h.